Detection of DNA damage

ABSTRACT

The health condition of a living organism is detected by electrochemically analyzing samples from selected areas of the body of said living organism for elevated free levels of nucleotide excision products resulting from DNA or RNA damage.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 09/687,047 filed Oct. 13, 2000 now U.S. Pat. No. 6,548,252.

FIELD OF THE INVENTION

The present invention relates to improvements in diagnostic techniques,and more particularly to diagnostic techniques for detecting andidentifying DNA or RNA damage. The invention has particular utility inthe detection of cervical cancer and will be described in connectionwith such utility, although other utilities are contemplated, as will bediscussed below in detail, including detection of other cancers andother disease conditions, as well as health conditions brought out byexposure to environmental insults such as radiation, metals, smoke andsolvents.

BACKGROUND OF THE INVENTION

The current gold standard for detecting cervical cancer in women is theso-called “PAP Smear.” However, the reliability of PAP Smear testing,which relies upon a technician's observations, under the microscope, ofcellular morphology, may be compromised by technician fatigue and/orsubjectivity. Even very experienced technicians may misread a slide. Ifa false-positive is “called” or the slide results in an “uncertain”call, the physical may err on the side of the patient's safety, and callfor a hysterectomy (uterus removal) or total hysterectomy (uterus andovary removal). This, results in the patient taking a lifetime dose ofhormone replacement medications to keep the body in balance. Of course,the failure to identify “a pre-cancerous condition” could lead to aneven more disastrous result to a patient.

OBJECTIVES OF THE INVENTION

It is thus a primary objective of the invention to overcome theaforesaid and other disadvantages of the prior art. Another objective ofthe invention is to provide an analytical technique for detecting anddiagnosing disease conditions, as well as health conditions due toexposure to environmental conditions, by detecting and identifying DNAor RNA damage markers. Another more specific objective of the inventionis to provide a reliable, totally objective analytical technique fordetecting cancer.

BRIEF DESCRIPTION OF THE INVENTION

In order to effect the foregoing and other objectives, the presentinvention provides an analytical technique for detecting cancer or otherdisease and/or health conditions based on measurement of free levels ofnucleotide excision products resulting from DNA or RNA damage, such as8OH2′dG-8 hydroxy 2′ deoxyguanosine; O6MG—O-6-methylguanine;2dG—2′deoxyguanosine; 7MG—7-methylguanine; 8NG—nitroguanine;8OHG—8-hydroxyguanine; 8OH2′dA—8-hydroxy-2-deoxyadenosine;8OHA—8-hydroxyadenine; 5OH2′dCy—5-hydroxy 2′deoxycytidine;5OHU—5-hydroxyuracil; 3NT—3-nitrotyrosine; or 3 CIT—3-chiorotyrosine inbiological samples from selected areas of the body. More particularly,the present invention is based on the hypothesis that specific areas ofthe body are semi-isolated in-situ biochemical environments fornucleotide excision products such as 8OH2′dG or other nucleotideexcision products, and that the levels of such free 8OH2′dG or othernucleotide excision products in the semi-isolated environment magnifythe combined defects of DNA or RNA damage and repair mechanisms. By wayof specific example, the effective damage and repair rate increases inDNA in cancer, or pre-cancerous cells, has been found to be magnified byaccumulation in the extra-cellular matrix in selected areas of the bodyof 8OH2′dG. For example, as applied to cervical cancer, the cervix hasbeen found to be a semi- isolated in-situ biochemical environment whichmay be accessed through cervico vaginal lavage sampling. Thus, anobjective analytical technique for determination of cervical cancer isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

For a further understanding of the nature and objectives of the presentinvention, presence should be had to the following detailed descriptiontaken in conjunction with the accompanying drawings wherein:

FIG. 1 is a schematic view of one form of sample separation and analysissystem in accordance with the preferred embodiment of the invention;

FIG. 2 is a side elevational view, in cross section, showing details ofa preferred form of sample preparation column useful in accordance witha preferred embodiment of the present invention;

FIG. 3 is a side elevational view, in cross section, of a separationand/or testing column controlled as an electrochemical cell useful inaccordance with a preferred embodiment of the invention;

FIGS. 4 and 5 are a series of graphs showing the current over time of anelectrochemical analysis for 8OH2′dG and O6MG, respectively, in cervicovaginal lavage samples in accordance with the present invention; and

FIG. 6 are coulometric electrode array system patterns of PAP smearsamples in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION

Further understanding of the features and advantages of the presentinvention will be had from the following detailed description of theinvention, which illustrates the electrochemical analysis of 8 hydroxy2′deoxyguanosine (8OH2′dG) in cervico vaginal lavage or PAP smear swabsamples. Analysis is accomplished using an electrochemical analyticalsystem made in accordance with and following the general analyticalprocedures disclosed in PCT Application No. PCT/US98/22275, and asdiscussed in “A Carbon Column Based LCEC Approach to Routine8-Hydroxy-2′-Doxyguanosine Measurements in Urine and Other BiologicalMatrices” (Bogdanov MB, et al. Free Rad. Ciol. Med. 27, 1227-1248,1999).

The conventional hypothesis of hydroxyl radical DNA damage and repairand excretion of the hydroxylated adduct 8OH2′dG is that a hydroxylradical reacts with the DNA causing hydroxylation of the deoxyguanosineat the 8 position. The damaged segment is then either excised byglycolysis as the 8Ohgua or by endonuclease excision as the8OH2′dG5′monophosphate. The monophosphate is then dephosphorylated andthe 8OH2′dG is rapidly excreted from the cell. The 8OH2′dG excreted tothe extracellular matrix is then cleared rapidly from the body primarilyvia the kidney and excretion in the urine. 8Ohgua can also be producedas a result of attack on RNA and glycolysis.

Studies performed with the technologies described above have confirmedcertain basic elements of this hypothesis as follows:

1. Dialysis studies of 8OH2′dG in extracellular matrix with probesplaced in brain and muscle of rat and mouse to measure the rate ofproduction and excretion compared with the free levels inside thecellular material confirm that excretion from the cells is rapid.Similarly, comparison of CSF, Plasma and urinary levels of ca 1:10:2000in ca 100 ALS and control subjects confirm that the rate of clearancefrom the body is high. Studies of saliva or sweat vs. urine levels andstudies showing highly elevated levels in plasma kidney dialysispatients confirm that urinary excretion is the primary mode of removal.

2. Studies of urinary levels of 8OH2′dG within individuals over diurnalweekly, monthly and yearly intervals indicate that the rate of DNAdamage and repair is highly constant and characteristic of anindividual. Similarly, studies of siblings and parents which show asignificantly closer agreement of sibling values indicate that the rateof DNA damage is strongly determined by genetic factors.

Other studies, however, have indicated some basic difficulties with thesimplistic model of damage and repair which have relevance to the use ofDNA damage markers as diagnostic tools.

1. The simple model of increased production of hydroxyl radical leadingto increased DNA damage is incomplete.

Comparison of total body rate of production of hydroxyl radicalestimated by salicylate spin trappings showed no correlation in normalindividuals, ALS patients and Freidrich's Ataxia (“FA”) patients ofurine or plasma levels of 8OH2′dG. Nor was there any group elevation oftotal body hydroxyl radical production in FA or ALS although the 8OH2′dGlevels were increased in CSF plasma or urine by 25-30%.

Urine levels of 8OH2′dG were significantly increased in individualsexposed to arsenic or o-toluidine or aniline although these materialsplay no direct role in the increase of hydroxyl radical production.

These studies suggest that conformational changes in DNA induced byexogenous or indogenous adducts or changes in sub cellular structures inproximity to DNA play a stronger role in increasing the rate of DNAdamage than overall hydroxyl radial production. Also supporting thisconcept are studies I have performed showing no effect of simpleantioxidants such as Ascorbate or Tocopherol on the levels of 8OH2′dG.

2. Relevant to the use of DNA damage rate measurements to cancerdiagnosis I have observed that in C. Elegans culture there is a burst of8OH2′dG production during the stage of rapid replication of the gonadalcells. In similar experiments I observed that a toxin (3 nitro propionicacid, 3NP) increased the rate of DNA damage during replication. When thetoxin was removed after exposure the rate of damage was still maintainedat a higher level indicating that the initiation of a higher rate ofdamage creates a biochemical state or feed back that persists beyond thetime of the applied insult.

3. It has also been observed in human controls, ALS patients and FApatients and in C. Elegans culture that the levels of 8OHS′dG thatremain in the DNA (measured by extracting and hydrolyzing the DNA) areonly weakly correlated with the rate of output of the 8OHS′dG. Thisindicates that both damage and repair are up regulated simultaneouslyand thus that measures of the free levels of excised DNA damage productprovide a much more robust indicator of disorder related DNA damageprocesses.

4. In situations where there is a high rate of damage and repair theconventional sequence of excision of the 8OH2′dG phosphate,dephosphorylation of the 8OH2′dG and excretion of the 8OH2′dG does nothold. In rapid cell replication in C. Elegans, in 3NP insulted C.Elegans and in Cervix cancer cells (as described below) the 8OH2′dGphosphates are directly excreted.

5. Sampling specific to the site of insult is of considerableimportance. In studies of smoking related increases of urinary 8OH2′dGlevels, I have found a statistically significant increase of only 11% ina cohort of ca 200 smokers vs. ca 300 control non-smokers. However, thefree levels of 8OH2′dG in the extra-cellular matrix from pharyngealswabs of smokers vs. non-smokers are elevated by a factor of 3×-4× whennormalized against the cellular metabolite of tyrosine, 4-hydroxy phenyllactic acid.

The synthesis of these studies and observations leading to the approachto cancer diagnosis is as follows:

1. When a cell begins extensive abnormal replication the changes in theconformation of the DNA during mitosis make it more susceptible to freeradial damage and both increase the levels of excised damage productsand change the nature of those products.

2. Sample sites which are semi-isolated in situ from physiologicaltransport and excretion, reflecting the extracellular matrix around theaffected cells will show highly increased levels of the repair productsrelative to the damage products remaining.

3. Levels of the repair products can be normalized against othercellular metabolites that are excreted to the extracellular matrix frommetabolic processes that are not materially affected by free radicaldamage.

WORKING EXAMPLES

Cervico vaginal lavage samples were centrifuged on acquisition toseparate the exfoliated cells and the supernatant containing theextracellular matrix. PAP smear swabs were vortexed in a normal salinesolution and centrifuged to separate the exfoliated cells andextracellular matrix.

Analysis for 8OH2′dG in supernatants was accomplished in accordance withthe teachings of PCT/US98/222275 using electrode preparation and sampleconcentration protocol described in Bogdanov et al, supra. Analysis for8OH2′dG phosphates was accomplished by treating a subaliquot of thesupernatants with alkaline phosphatase following the last stage of theDNA hydrolysis protocol described in Bogdanov et al, and determinationof the 8OH′2dG phosphate level by difference between the alkalinephosphatase treated and the non-treated aliquot. Chiorotyrosinemeasurements of subaliquots of the supernatant were made according tothe teachings in PCT/US98/22275 following the protocols in Bogdanov etal.

The process for determining appropriate normalizers and markers ofsample integrity was to prepare and analyze extracts of the supernateand cells from cervico vaginal lavage and PAP smear samples, plasma redblood cell and leucocyte samples, buccal cell samples and induced sputumsamples following the procedures in Kristal et al “Simultaneous Analysisof the Majority of Low-Molecular Weight Redox-Active Compounds fromMitochondria,”Biochem. 263, 18-25(1998). Typically 600 peaks of redoxactive compounds are isolated from such preparations. The varioussamples were compared against themselves and against authentic referencestandard mixtures primarily to search for peaks or unknown compoundswith the following characteristics:

1. The peaks should be unique to cervico vaginal lavage or PAP smearsamples in order that it not be compromised by inclusion of othercellular or biological material inclusion.

2. The peaks should be present in the supernate and not in the cellularextracts or at significantly higher relative concentration than thecellular extract in order to reflect a similar high rate of excretionfrom the cells as 8OH2′dG.

Secondarily, the patterns of cervico vaginal lavage and PAP smearsamples were compared against urine and blood samples to determine thelevel for compounds that are highly elevated in blood, e.g. glutathioneand urine, e.g. uric acid such that levels of these compounds indicatecontamination and effects on 8OH2′dg levels could be esablished.

Of 16 peaks in the patterns meeting the criteria 1 and 2 Tyramine wasidentified. The other 15 peaks meeting the criteria are currently ofunknown structure although they are also candidates for normalization.Levels of uric acid and glutathione in cellular material and supernatefrom cervico vaginal lavage and PAP smear samples established levels ofca 1000 ng/ml of uric acid and 50,000 ng/ml of glutathione below whichno measurable impact on the 8OH2′dG would be expected. A typical exampleof the comparison for known compounds among cell types is shown in TableI.

The carbon column switching instrumentation and the arrayinstrumentation were combined by placing an 8 channel series sensor onthe output of the first analytical column of the carbon column switchingapparatus and a second 8 channel series sensor on the output of thesecond analytical column of the carbon column switching apparatus. Thisallowed the simultaneous determination of the uric acid glutathione andtyramine on the output of the first analytical column and the carboncolumn trapping, elimination of interferences and detection of 8OH2′dGand Chlorotyrosine on the second analytical column.

FIGS. 4 and 5 show typical responses for 8OH2′dG and O6MG in PAP smearsamples. The figures illustrate the detection of 8OH2′dG in all samplesand the limitation in detecting O6MG in some.

FIG. 6 illustrates the CEAS patterns observed from cellular andsupernatant preparations. These were compared against each other andagainst similar patterns of urine, plasma, whole blood and buccal cellsto determine normalizing compounds and indicators of sample quality.

The results of the analytical protocols and concepts applied to 51cervico vaginal lavage samples from 51 patients with various diagnosesand two normal PAP smear samples are shown in Table I.

Table II presents the data on 8OH2′dG, O6MG vs. an abbreviateddiagnostic categorization ranked by tyramine divisor and sample weightor weight estimated from protein. CVL and PAP smear samples are rankedseparately. Initial studies of vaginal vs. cervical swabs indicatedhigher levels of tyramine in the former. It is thus likely that tyramineis not an optimum normalizer since there are several other peaks thatmeet the criteria but have not been chemically identified. Table IIIpresents preliminary data on relationships among free and DNA levels of8OH2′dG, in the cervix and urine and plasma. There are some limitationson the data quality affecting interpretation. Precision on small samples(less than 2 mg) is only +/−220-30%. Storage histories are notcompletely documented and there are uncertainties associated with anystudy of a new matrix. However, some observations are indicated.

TABLE I CASE Compound 00000001.ora 00000003.ora 00000006.ora00000008.ora 00000009.ora 00000014.ora 00000016.ora 00000017.ora00000024.ora 00000029.ora 00000033.ora ng/ml CL BUC MUC BUC MUC BUC MUCCL BUC MUC CL CL CL BLOOD ca1:15 CL 2 HPAC 0.63 0.40 0.20 0.09 0.25 0.620.24 294.78 3.23 1.96 3 MT 0.13 0.19 0.18 0.08 0.12 0.17 0.11 0.22 1.390.33 3 OHKY 0.20 0.31 0.16 0.24 0.15 0.16 0.48 0.65 0.52 3 OMD 0.76 0.310.39 0.44 0.32 0.39 0.51 0.53 1.31 0.39 4 HBAC 0.26 0.44 3.26 2.94 3.856.78 0.47 1.83 0.72 0.19 4 HPAC 36.33 43.35 8.45 8.27 9.35 1.49 244.444.48 658.94 0.99 24.89 4 HPLA 120.24 92.60 31.05 28.88 24.37 4.67 66.4137.64 665.94 0.75 55.39 5 HIAA 0.47 0.22 0.07 0.24 0.08 0.10 0.48 0.150.19 5 HT 0.10 0.11 0.11 1.13 0.11 0.26 0.79 0.19 5 HTP 0.06 0.11 0.160.19 0.06 0.24 0.54 0.16 0.13 AM 6.48 0.08 0.07 0.10 0.06 0.25 1.70 0.1113.69 0.11 0.40 ASC 0.62 0.13 1011.01 0.35 0.16 416.21 5.77 1.14 0.62CYS 70.29 72.53 54.53 82.05 70.29 76.22 88.99 45.98 49.09 21.61 48.44 DA21.93 6.66 0.94 3.27 14.19 1.25 2.01 0.38 3.33 0.43 0.41 DOPAC 0.11 0.110.12 0.09 0.13 0.25 0.10 1.40 0.37 0.22 G 12.52 8.62 3.44 237.98 19.463.63 61.18 89.06 1.38 1.27 86.94 GR 12.47 32.41 6.45 30.36 3.53 11.7559.25 5.07 16.22 11.61 130.11 GSSG 3.10 111.76 25.78 24.08 42.78 37.48193.42 27.37 2.12 1901.78 130.07 HGA 0.19 0.11 0.25 0.14 0.16 0.22 0.250.51 0.23 HVA 0.23 0.16 0.09 0.10 0.07 0.14 0.15 0.18 0.10 0.38 0.57 HX24.75 39.98 8.10 619.81 48.07 74.60 461.74 152.57 2441.70 2445.10 112.92KYN 1.03 0.09 24.39 1.63 0.29 0.07 5.09 0.48 28.95 0.30 9.74 LD 1.232.43 0.59 0.57 1.29 0.56 0.50 0.65 3.26 0.67 0.91 MEL 0.92 0.92 1.104.49 4.81 0.16 1.81 0.22 0.62 0.49 1.02 MET 42.88 0.82 4.21 441.49 43.941.27 464.70 39.17 73.65 831.72 877.84 MHPG 0.46 0.51 0.55 0.42 0.44 0.480.61 0.95 0.66 0.40 MN 1.22 0.17 0.65 0.30 0.28 0.20 0.39 0.26 0.25 0.571.28 NA5HT 0.14 0.17 0.11 0.14 0.21 0.17 0.17 0.18 0.12 NE 0.40 0.100.33 0.19 0.36 0.26 0.32 0.26 0.22 NMN 0.30 0.51 0.28 0.32 0.49 0.290.50 0.37 0.34 0.32 3.30 TPOL 8.59 6.11 7.77 9.11 11.33 13.89 16.4736.25 1.19 1.41 28.63 TRP 186.72 27.57 7.44 240.92 47.34 5.07 53.6555.25 3904.06 523.63 237.29 TRYPT 0.15 1.19 0.61 0.83 82.32 0.43 8.190.74 13.85 0.50 277.80 TYR 266.00 551.51 251.17 717.54 98.95 140.30214.95 227.17 7411.08 1545.97 639.34 TYRA 579.81 10.61 0.19 0.90 525.822.03 772.19 330.77 35.39 0.15 1088.90 URIC 695.51 703.83 3209.99 221.6048.09 580.64 229.01 405.36 314.90 1992.77 371.60 VMA 0.44 0.43 0.12 0.260.10 0.27 0.50 0.17 1.12 XAN 73.44 235.05 16.67 107.60 16.00 53.931458.70 58.20 3669.12 343.39 45.54 XANTHOSINE 19.29 83.74 1.75 37.0020.15 21.78 23.70 16.64 1461.96 151.37 34.65 NORMALIZERS 8 OHdG pg/ml11.2 3.33 2.22 8.47 10.8 4.9 17.39 2.42 635.48 0.32 1.61 80 h/lyrx 1004.21 0.60 0.80 1.10 10.91 3.49 8.09 1.07 8.57 0.02 0.25 8 oh/cysx 10015.93 4.59 4.06 10.22 15.36 6.43 19.54 5.26 1273.76 1.48 3.32 8 oh/tyrax1000 10.32 313.84 11684.21 9411.11 20.54 2413.79 22.52 7.32 17956.492133.33 1.48

TABLE II CASE CODE 8 OH pg/gm CVL SAMPLES 8 OH*1000/tyr DIAG CODE cal byprotien 8 OH/O6 MG ESA006 17652.22 C 108934 0.12 ESA041 5198.99 C 87132ESA0447/21 3827.08 C 38897 ESA0447/16 3452.49 C 42132 0.24 ESA0512441.30 C3 44150 ESA037 1881.84 A,D,R 31988 ESA011 1800.00 C NA ESA0481373.63 A,D,R 12012 ESA033R3 940.82 A,D,R 18821 1.33 ESA025CVL 635.73A,R 28812 ESA033R1 564.89 A,R 15168 ESA034 513.59 A,D,R 12997 0.92ESA030 513.55 A,R 19281 ESA043 510.68 A,R 12113 0.77 ESA038 502.68 A,R23881 ESA040 215.30 A,R NA ESA050 197.87 A,R 10687 1.92 esa032s 121.11A,D,R 12188 esa029s 74.25 A,R 2188 ESA035s 38.50 A,D,R 1722 ESA018CVL35.74 A,R 3221 ESA022CVL 27.73 A,R 3324 1.01 ESA004 22.53 A,R NAESA019CVL 22.05 A,D,R 1922 ESA003 20.53 AR 1334 ESA023CVL 20.49 A,R 1987ESA001 19.31 A,D,R 1332 ESA039 15.53 AR 1435 ESA005 7.33 A 397 ESA0363.79 A 433 esa027CVL 2.91 A 198 ESA047 1.56 N 163 GT10 ESA012 1.48 N 289ESA049 1.33 N 143 ESA020CVL 0.71 A 206 esa031s 0.71 N 134 ESA048 0.64 N289 ESA021CVL 0.81 A 189 7.44 ESA042 0.57 N 215 ESA045 0.45 N 127 GT10ESA026CVL 0.36 N 131 ESA024CVL 0.18 N 156 CASE CODE PAP SMEAR SAMPLES 8OH*1000/tyr DIAG CODE 8 OH pg/gm 8 OH/O6 MG CON1R1 0.92 N 368 7.2 CON21.03 N 442 11.2 CON3 0.83 N 293 14.1 CON4R1 0.72 N 528 NA CON1R5 endobrush 1.06 N 445 6.8 exo brush 0.94 N 502 NA vaginal padl 0.12 N 28 NACON5R1 2.44 N 308 11.1 CON4R3 0.98 N 122 4.3 CON5R3 1.87 N 354 7.5 WOO12.19 A, R 125 1.25 W002 4.1 A, R 507 0.73 W003 58.28 Hepatitis 3855 0.57W004 8.23 A, R 200 0.58 W005 2.19 N 75 1.58 W006 4.3 N, R 500 0.84 W0072.1 N 90 0.71 W008 27.49 N, HPV 361 0.55 W009 113.04 ?A, HPV 1300 0.45W010 0.9 7A ?5 1.68 W011 1.6 N, R 75 1.2 W012 1.8 N 85 1.48 W013 1.1 N75 1.2 W014 1.8 N, R 145 1.8 W015 1.9 N 199 4.4 W016 2.8 N, R 301 4.3W017 2.8 A, R 190 1.3 W018 3.12 A 412 1.9 W019 1.4 N 138 7.3 W020 1.87 A115 4.4 W021 4.12 N, R 543 1.7 W022 1.7 A 214 GT10 W023 56.9 N, HPV 32240.41 W024 2.1 A, R 341 3 W025 0.81 N 104 NA W026 23 N, R 122 5.7 W0272.8 A, R 107 4.4 W028 1.6 N 115 8.1 N—normal PAP, A—ASCUS, C—cancer,C3—CIN3, D—dysplasia, R—varios risk factors, HPV—PCR+ viral te NA—notanalysed, interference

TABLE III CVL SAMPLE DNA pm/um PLASMA Urine 8 OH*1000/ URINE ug/gm 8OH′dG/ pg/ml urine code tyra 8 OH′dG/creat 2′dG 8 OH2′DG esa110u 3.15esa003u 4.97 esa004u 4.57 esa005u 17652.22 14.48 5.81 31.90 esa012u5198.99 3.30 16.30 esa006u 2441.30 5.73 esa018u 1881.84 2.38 6.13 22.10esa019u 1373.63 1.71 esa020u 940.82 4.11 835.73 2.86 4.98 14.60 esa022u564.89 3.94 5.87 esa023u 513.55 1.79 esa024u 513.55 4.83 esa025u 510.662.19 esa026u 502.68 2.22 esa027u 215.30 5.38 esa029u 197.87 4.14 esa030u121.11 2.16 esa031u 74.25 1.64 11.80 esa032u 38.50 3.25 esa033au 35.743.05 esa033bu 27.73 2.40 3.44 esa034u 22.53 2.15 3.38 15.30 esa035u22.05 3.04 esa036u 20.53 2.15 5.12 16.40 esa037 20.49 5.45 4.76 esa038u19.31 3.95 esa039u 15.53 3.55 esa040u 7.33 4.24 4.16 11.50 esa041u 3.792.13 4.28 esa042u 1.56 3.64 esa043u 1.56 3.64 esa0447/16u 1.48 3.33 3.36esa0047/21u 1.33 3.71 esa045u 0.71 3.24 12.30 esa046u 0.71 6.69 17.80esa047u 0.64 1.81 esa048u 0.61 2.58 esa049u 0.57 3.29 esa050u 0.45 2.50esa051u 0.36 4.11 esa011u 0.18 2.50

Results

Free levels of 8OH2′dG in pg/ml were 1.61 (normal case), 2.42 (precursorcell case) 10.8-17.4 (dysplasia cases n=3) and 635.5 (cervical cancercase). The values corrected for dilution by various measures of totalprotein, amino acids or cell count, indicate initial concentrations inthe extracellular cervical environment of ca 80-110,000 pg/ml or −1000times the normal plasma levels of 10 pg/ml. This supports the hypothesisthat the cervix is in a biochemically isolated area. Ratios of8OH2′dG/2′dG in the DNA of exfoliated cells were only elevated byfactors of 2.1 and 4.8 in the Dysplasia and cancer cases respectivelyvs. the normal. This also supports the hypothesis that the effect ofincrease in both DNA damage and repair is magnified in the extracellularmatrix in the cervix environment.

It is also consistent with the intuitively less probable hypothesis thatthe DNA damage and repair is proceeding at a 20-100 fold higher rate inthe cervix. A weak correlation (r=0.431), driven by high values in thecancer patients between urinary and cervical levels, is also consistent.In the cases with repeat cervical samples, the values were consistentwithin an individual. Urinary 8OH2′dG values are highly individuallyspecific and the same may hold for the cervix.

The absolute levels of 8OH2′dG based on estimates of the amount ofextracellular matrix from protein values in cervico vaginal lavagesamples and actual weights of PAP smear samples are on the order of200-800 pg/ml or ca 20-80 times the levels of 10 pg/ml observed inplasma (2). This supports the hypothesis and necessary diagnosticrequirement that the cervix is a semi-isolated in-situ area with respectto transport and excretion of DNA damage products.

The very slight not statistically significant increase in DNA damageproducts in the DNA itself supports the hypothesis that both damage andrepair are up regulated and that the effect is magnified in theextracellular matrix.

While uncorrected 8OH2′dG concentrations are statistically higher in thecancer patients and patients at risk than in controls the use ofTyramine as a normalizer reflecting sample dilution and acquisitionfactors of the extracellular matrix is effective in compressing thescatter of the data.

Tyramine normalized levels of 8OH2′dG in all control samples and in twocases of findings of Abnormal Squamous Cells of Uncertain Significance(ASCUS) are all below the level of 1.6 pg 8OH2′dG/ng Tyramine. Allsubjects with identified cervix cancer and two with a high degree ofsuspicion of cancer are about 16000 pg 8OH2′dG/ng Tyramine. Patientswith intermediate possibly precancerous problems and risk factors have8OH2′dG/Tyramine ratios that are intermediate between control and cancerand may predict an enhanced degree of risk of an early cancer that willmanifest subsequently.

The presence of measurable 8OH2′dG phosphate as a significant portion ofthe total 8OH2′dG occurs in all the cancer cases and in three of thecases with significant risk factors and initial per cancerousindicators.

Chlorotyrosine is found in seven cases and may in the case of ESA051indicate a significant level of infection as well as or instead of anearly cancer lesion.

Thus, the invention and concept provides an index of 8OH2′dG/Tyraminethat separates cervical cancer subjects from controls by a minimumfactor of 1000 and gives intermediate values for various degrees of riskand findings of abnormal or precancerous cells or lesions. A uniquemarker of excretion of a portion of 8OH2′dG as the phosphate is found inall cancer subjects and may provide the first indicator of theculmination of risk and insult factors in an early cancer in theindividuals with high 8OH2′dG/Tyramine ratios. The ability to commeasuremarkers of inflammation and infection provides additional diagnosticdiscrimination between risk factors and actual development of acancerous lesion.

Several possible candidates for dilution normalizing compounds also wereidentified from application of a protocol for analysis of the majorityof oxidizable compounds in the cervical lavage supernate. Tyramine(TYRA) notably appears to be an attractive normalizer:—it is a uniquelyelevated product of cervical cellular metabolism relative to othercellular material (Buccal cells, lymphocytes, alveolar and haryngeallavage, brain and muscle dialysates); it is excreted rapidly to theextracellular matrix; and it is co-determinable with 8OH2′dG with thetechnology employed. For the six test cases, the mole ratios of8OH2′dGx1000/TYRA were: 1.48 (normal case), 7.34 (precursor cell case),19.3-22.5 (Dysplasia cases n=3) and 17956 (Cervical cancer case).

Thus, there appears to be a correlative decrease in 8OH2′dG levels withincreased coQ10 levels in plasma, or in the local in situ cervical area.Accordingly, the analytical strategy of measuring both free and DNAlevels of 8OH2′dG in conjunction with the coQ10 also provides a basisfor discriminating between changes in the rate of oxidative damage andchanges in the rate of repair associated with the coQ10 levels.

The invention is susceptible to modification. For example, analysis ofstool samples for the presence and level of 8OH2′dG and 8OH2′dGphosphates (which typically do not occur above about 0.1 pg/gm in normalfeces) both as timed or total output and normalized against levels ofthe tyrosine metabolite gentisic acid may be employed as a diagnostic ofcolon or lower bowel cancer; analysis of saliva for elevated levels of8OH2′dG may be employed to provide an early test for esophageal orthroat cancer; analysis of urine or semen for elevated levels of 8OH2′dGmay be employed to provide an early test for bladder or testicularcancer; pharyngeal or nasal swab, alveolar or bronchial lavage samplesfor analysis of 8OH2′dG normalized against levels of 4-Hydroxy Phenyllactic acid may be employed as a diagnostic of pharyngeal, nasal,bronchial or lung cancer respectively; analysis of foliated urithelialcells in timed culture for 8OH2′dG levels normalized against cell numberor weight may be employed for diagnosis of bladder or prostate cancer;analysis of menstrual blood for 8OH2′dG leads may be employed for earlydetection of ovarian cancer; analysis of phlegm for elevated levels of8OH2′dG may be employed to provide an early test for lung cancer; andanalysis of throat swabs may be employed to provide an early test forthroat or esophageal cancer.

While not wishing to be bound by theory, it is believed that 8OH2′dGfree level measurements represent the combined effect of the rate of DNAdamage and repair caused by disease conditions or environmental insultsin a living organism. Essentially, it is believed that the whole bodyrate of production of 8OH2′dG is:

-   -   (a) constant in an individual over time in the absence of        disease or environmental insult;    -   (b) genetically determined;    -   (c) not related to whole body rate of Hydroxyl radical        production;    -   (d) increased by environmental insult from putative DNA adducts        (e.g. pyrenes, Toluidine Arsenic, etc.);    -   (e) increased in several neurodegenerative diseases;    -   (f) assessed equally well by spot and 24 hour urines;    -   (g) not affected by antioxidants tocopherol and ascorbate; and    -   (h) not affected by diet and converted (via HCl and glycolase)        to 8OH2′dGua in the stomach.

The clearance rate from cells and the body as a whole is believed to bevery rapid. Normally clearance is via urine but also may be by salivaand sweat. If rates are accounted for there are compartmentalcorrelations among plasma and urine and saliva and DNA damage levels inleucocytes. Typically, ca. 30% of the whole body 8OH2′dG output is fromthe brain.

Compartmental correlations can be used to determine specific organinvolvement. For example, in C. Elegans culture there are bursts ofproduction during rapid mitosis. Isolated Mitochondrial preparationsalso produce 8OH2′dG in respiration. The relationships among differentadducts are different in rapid mitosis, stable cell number cultures andmitochondrial cultures. Levels in DNA are only weakly correlated withrate of production.

Physiologically or transport isolated reservoirs concentrate theproducts and magnify the effects of DNA damage and repair, e.g. althoughI have measured only an 11% increase in urine levels of smokers, I havemeasured a 400% increase in pharangeal swab samples.

Thus, it is believed that rates of DNA damage are primarily affected by:(a) factors causing changes in the conformation of the DNA (mitosis,binding of endogenous and exogenous adducts, transcription); and (b)increases in free radical production in subcellular structures in closedimensional proximity to the DNA (lipid bilayer, nuclear cytosol). Thedamage and excision product profiles differ for mitochondrial andnuclear DNA and by mechanism of damage. These observations are forcervical cancer, but they are believed equally generalized to lung,lower bowel/colon, prostate, bladder cancer, etc. as follows:

1. The cervix and the extracellular matrix of the cervix is an in situsemi-isolated physiological area with respect to transport of excretedcellular metabolites.

2. The product 8OH2′dG of the combined process of DNA damage and repairis concentrated in the extracellular matrix of the cervix and magnifyand reflect the effect of these processes more than the levels of thedamage products remaining in the DNA.

3. Correlative or normalizing markers for control of sample dilution andacquisition effects can be selected from metabolites that are (a) notaffected by free radical processes, (b) excreted from the cells at arapid rate, or (c) unique or predominant to the metabolism of cervicalcells.

4. The rate of production of DNA damage/repair products will beincreased when the cells in the cervix are (a) undergoing rapiddivision, (b) exposed locally to increased free radical insult as frominfection or inflammation, (c) exposed to increased levels of DNAconformation modifying adducts from endogenous processes (metabolicshifts) or exogenous sources (bacterial, or viral infections and/orenvironmental toxins).

5. The combination of the factors increasing the rate of DNA damage willconstitute a risk factor for cancer which will be reflected in higherlevels of DNA damage and repair product 8OH2′dG in the extracellularmatrix.

6. The magnitude of the different mechanisms of DNA damage will differand rapid cell division will predominate. Thus, rates of DNA damage incancer cases and extracellular levels of 8OH2′dG will be significantlyhigher than in cases of ASCUS or with dysplasia or other risk factors.

7. The relative levels of DNA damage and repair products will differ fordifferent mechanisms. Cancer cells will excrete a significant portion ofthe endonuclease excised 8OH2′dG as the phosphate or monophosphate, andexcise a higher proportion of the damaged base by glycolase excisionresulting in elevated levels of 8OH2′dGua in the extracellular matrix.Cancer cells will also preferentially excrete elevated levels of5-Oxocytidine and reduced levels of 8-nitro guanine.

8. The absolute and relative levels of 8OH2′dG, 8OH2′dG Phosphates,8OH2′dGua, 8 nitroguanine and 5 Oxocytidine will constitute a profileindicating the presence of cancer independent of the cytological resultsof the PAP smear and levels of risk factors.

9. The absolute and relative levels of DNA damage products will providean early indication of cells that are in a precancerous state.

10. Co-determined markers of other free radical damage products willserve to further differentiate the nature of and extent of damage fromother risk factors to the rate of DNA damage. Notably chlorotyrosinewill serve as an indicator of the effect of inflammation or infectionand nitrotyrosine as a normalizer for peroxynitrite DNA damageprocesses.

It is thus seen that:

1. The levels of 8OH2′dG in cervical vaginal lavage normalized byprotein or in PAP smear samples from controls normalized by weight areca 250-500 pg/ml of extracellular matrix or ca 25-50 times plasma. Thisis in contrast to the free extracellular levels in muscle, brain, liver,etc. which are on the order of ca 1-3 pg/ml. Therefore, the cervix isseen as semi-isolated with respect to metabolite transport. In cancercases where the levels of 8OH2′dG in the extracellular matrix are ca 100times higher than in controls the levels in DNA from the exfoliatedcells are only increased by 35% and the difference is not statisticallysignificant. So the concentration of the products in the semi-isolatedarea magnifies the effect of the DNA damage rate.

2. Using pattern generating techniques to compare cell extracts andextracellular matrix from CVL, PAP smear, buccal cell and alveolarlavage samples and also plasma, red blood cells and urine, the followinghas been observed:

In CVL and PAP smear samples 23 candidate peaks were over 10× moreconcentrated in the extracellular matrix, 3 where highly elevated orunique in the CVL/PAP smear samples over all other types. Tyramine wasidentified as one of these which should be minimally affected by freeradical processes and was selected as a sample acquisition normalizer.Note on an operational basis these studies also served to provideacceptable levels of sample quality indicators, e.g. uric acid for urinecontamination and glutathione for blood inclusion.

3. The ratio of 8OH2′dG/tyramine seems to indicate that the rate ofdamage is higher with cancer and with various risk factors which mayinclude HPV infection, other STD's, multiple partners, hisuitism,endocrine abnormalities, and various equivocal findings from colposcopy.

4. There appears to be a gradation in the increase in rate of DNA damagethat is generally in the same direction as increase in risk factors.

5. Even if any of the assumptions are wrong, the rates of local DNAdamage in the cervix in the cancer cohort is much higher than in thecontrols or at risk cohort. Thus, a viable cancer screening testsubsists.

6. Some 8OH2′dG phosphate is always seen in cancer. Thus, when it isseen in subjects with high rates of DNA damage and other risk factors itis likely that they actually do have cancer, but it just hasn't advancedto a point where it can be conventionally detected yet. The8OH2′dGua/8OH2′dG ratio was 20 fold higher in two cancer patients thanin 8 controls and an algorithm based on ratio and absolute levelseparates the categories by a factor of 20,000.

The invention is susceptible to modification. For example, the abovetechnique may be employed to assay free levels of the primary oxidativeDNA excision product 8-hydroxy-2′-deoxyguanosine (8OH2′dG) and samplenormalizing compounds in pharyngeal swabs, induced sputum, bronchial andalveolar lavage using carbon column switching liquid chromatographyelectrochemical detection (LCEC) as above described. Preliminary workshows three fold elevations of 8OH2′dG in sputum and pharyngeal swabs insmokers. The absolute levels of 8OH2′dG are 10-30 times greater than inplasma which supports an initial hypothesis that the respiratory tractis a semi-isolated physiological transport limited area in which theprocess of DNA damage and repair will be magnified. In the cervix, asimilarly semi-isolated physiological area, cancer patients shown2000-10000 fold elevations of 8OH2′dG over controls. Thus, there isprovided a diagnostic or risk assessment biochemical test forrespiratory lesions.

As before, and while not wishing to be bound by theory, the overallhypotheses is that:

1. The respiratory tract is a semi-isolated physiologically transportlimited area in which the effects of DNA lesion repair will bemagnified.

2. Increased DNA damage will be coupled with up regulated repair andthus the free levels of repair products in the extracellular matrix ofthe respiratory tract and thus will be more indicative of risk orpresence of disease than the levels in the DNA itself.

3. The levels and nature of DNA and RS damage products will differ amongprocesses of DNA ligand formation, increased reactive species product,inflammation/infection and rapid cell mitosis, necrosis and apoptosisand thus provide a route to diagnosis or risk assessment.

The assay of 8OH2′dG and potential sample normalizers in pharyngealswabs, induced sputum and bronchial alveolar lavage samples, urine,plasma and leukocytes from cohorts of controls, individuals with variousrisk factors and individuals with respiratory tract lesions usingexisting technologies permits the determination of the correlations andcompartmental relationships among the different sample types and thedifferent diagnostic categories for 8OH2′dG and sample normalizers. Thisin turn permits evaluation of correlative and process relevant reactivespecies markers: 8-Hydroxyguanine, 8-Hydroxyguanosine (glycolaseexcision and RNA damage markers); 7-Methyl guanine (DNA methylationmarker); 3-Nitrotyrosine and 8-Nitroguanine (peripheral and DNA reactivenitrogen markers) and 3-Chlorotyrosine (infection or inflammationmarker).

The invention also provides strong evidence for concurrent methylationincreases in N methyl and NNdimethyl Serotonin (NM5HT NNM5HT) and7Nmethyl Guanine (7MG) in plasma and CSF and reduction in the oxidativeDNA damage marker 8-Hydroxy 2′Deoxyguanosine (8OH2′dG) in stroke andischemia in humans. More particularly employing the analysis techniquesof the present invention shows a reduction in CNS production of theoxidative DNA damage product 8-Hydroxy-2′-deoxyguanosine (8OH2′dG) andan increase in the production of methylated compounds in a cohort ofstroke patients and a similar reduction of 8OH2′dG levels in animalstroke models. Thus, elucidation of the biochemical mechanisms ofmethylation and oxidative free radical damage to DNA and other cellularstructures in stroke and related disorders, and the determination of therelationships of the biochemistry to stroke management, provides a rapidsingle assay technology for methylated, reactive oxygen and nitrogenspecies (ROS, RNS) markers in clinically accessible samples and samplesrelevant to mechanistic studies in animal models. This provides forclinical management of and the evaluation of pharmacologicalintervention in stroke.

Preliminary work with purine specific carbon column switching techniquesas above described also demonstrates elevations of the DNA hydroxyradical damage marker 8-hydroxy-2′-deoxyguanosine in a number ofneurodegenerative diseases (NDD). Progressive increases with diseasehave been shown in, e.g. amyotrophic laterial sclerosis. That is to say,preliminary work with carbon column switching LCEC technology inaccordance with the present invention has shown that the ROS DNA damagemarker 8-hydroxy-2′deoxyguanosine (8OH2′dG) is elevated in Parkinson'sDisease (PD) (urine, plasma, and CSF), Alzheimer's Disease (AD) (plasmaand urine, Huntington's Disease (HD) (urine), Freidrich's Ataxia (FA)(urine plasma) and that it is elevated and progressive with time inAmyotrophic Lateral Sclerosis (ALS) (urine plasma and CSF) but not inmyopathies. Secondary evidence from the patterns of chromatogramsdetermining 8OH2′dG indicate differences in unidentified purine adductsin urine plasma and CSF among the neurodegenerative disorder (NDD)categories. Again, while not wishing to be bound by theory, the overallhypothesis is as follows:

1. DNA damage products resulting from processes ofmethylation/ROS/RNS/RCLS are different among controls and NDD.

2. Ligand of endogenous/exogenous compounds with DNA play a role inincreased DNA damage.

3. Excision and repair processes differ among controls and NDD.

4. The sites of attack of RS on DNA differ among controls and NDD.

As mentioned supra, other DNA and RNA damage markers and free radicalmarkers may be assayed as indicators for the presence of or risk ofvarious health disorders.

Table IV below lists preliminary conditions developed for DNA damagemarkers and free radical markers established for the purpose ofestablishing approximate levels of the analytes of interest. Urine,plasma and cervical extracellular matrix values are presented for pooledcontrol samples. The basic sensitivity of all procedures is from 10-50pg/ml (500-2500 fg on column).

TABLE IV ug/gm pg/ml creatinine pg/gm A B/D C1/C2 G1/G2 Plasma UrineCervical 8 OH2'dG 10.3 3.86 200 O6 MG 20% MEOH(B1)  6% An(B2) ADN 1/1A/B  0.5 0.32  44 2'dG/7 MG 10% MEOH 3% An(B1)  7% An(B2) ADN 2/1 A/B500/3 ND/0.6 2000/15 8 OHG  1% MEOH(B1)  1% An(B2) ADN 1/1 B/B 26 9.83 80 8 NG 25% MEOH 4% AN(B2) 18% An(B1) NBA 1/1 A/B ND ND (?) 8 OH2'dA 8% MEOH(B1)  4% An(B2) DD 2/1 A/A ND 0.60  30 5 OH2'dCy  1% MEOH(B3) 1% An(B2) ADN 1/1 B/C ND ND  30 5 OHU  1% MEOH(B3)  1% An(B3) ADN 1/1B/C ND 0.40  56 3 NT/3 CIT 10% MEOH 3% AN(B1)  7% An(B1) NBA 2/1 B/B3.7/1.1 ND 20/10 MEOH - methanol An - acetonitrile; B1 - lithium acetate0.1 M pH 6; B2 - lithium phosphate 0.1 M pH 3; B3 - pentone sulfonicacid 0.1 M pH 4; Column Types: 1 - Tosobaas C18 ODS 80 TM; 2 - YMC C8Y02H1 carbon column; A - 4.6 mm d × 4.6 mm; B - 4.6 mm d × 8 mm; C - 4.6mm d × 13 mm; ADN—adenosine; DD—dodecane; NBA—nitrobenzoic acid; ND—notdetected

It is thus seen that the present invention provides an objectivereproducible test for cervical cancer and pre-cancer screening, as wellas for early detection of various other cancers and other healthconditions including heart disease and various degenerative diseases.

CASE Compound 00000001.ora 00000003.ora 00000006.ora 00000008.ora00000009.ora 00000014.ora 00000016.ora 00000017.ora 00000024.ora00000029.ora 00000033.ora ng/ml CL BUC MUC BUC MUC BUC MUC CL BUC MUC CLCL CL BLOOD ca1:15 CL 2 HPAC 0.63 0.40 0.20 0.09 0.25 0.62 0.24 294.783.23 1.96 3 MT 0.13 0.19 0.18 0.08 0.12 0.17 0.11 0.22 1.39 0.33 3 OHKY0.20 0.31 0.16 0.24 0.15 0.16 0.48 0.65 0.52 3 OMD 0.76 0.31 0.39 0.440.32 0.39 0.51 0.53 1.31 0.39 4 HBAC 0.26 0.44 3.26 2.94 3.85 6.78 0.471.83 0.72 0.19 4 HPAC 36.33 43.35 8.45 8.27 9.35 1.49 244.44 4.48 658.940.99 24.89 4 HPLA 120.24 92.60 31.05 28.88 24.37 4.67 66.41 37.64 665.940.75 55.39 5 HIAA 0.47 0.22 0.07 0.24 0.08 0.10 0.48 0.15 0.19 5 HT 0.100.11 0.11 1.13 0.11 0.26 0.79 0.19 5 HTP 0.06 0.11 0.16 0.19 0.06 0.240.54 0.16 0.13 AM 6.48 0.08 0.07 0.10 0.06 0.25 1.70 0.11 13.69 0.110.40 ASC 0.62 0.13 1011.01 0.35 0.16 416.21 5.77 1.14 0.62 CYS 70.2972.53 54.53 82.05 70.29 76.22 88.99 45.98 49.09 21.61 48.44 DA 21.936.66 0.94 3.27 14.19 1.25 2.01 0.38 3.33 0.43 0.41 DOPAC 0.11 0.11 0.120.09 0.13 0.25 0.10 1.40 0.37 0.22 G 12.52 8.62 3.44 237.98 19.46 3.6361.18 89.06 1.38 1.27 86.94 GR 12.47 32.41 6.45 30.36 3.53 11.75 59.255.07 16.22 11.61 130.11 GSSG 3.18 111.76 25.78 24.08 42.70 37.48 193.4227.37 2.12 1901.78 130.07 HGA 0.19 0.11 0.25 0.14 0.16 0.22 0.25 0.510.23 HVA 0.23 0.16 0.09 0.10 0.07 0.14 0.15 0.18 0.10 0.38 0.57 HX 24.7539.98 8.10 619.81 48.07 74.60 461.74 152.57 2441.70 2445.10 112.92 KYN1.03 0.09 24.39 1.63 0.29 0.07 5.09 0.48 28.95 0.38 9.74 LD 1.23 2.430.59 0.57 1.29 0.56 0.50 0.65 3.26 0.67 0.91 MEL 0.92 0.92 1.10 4.494.81 0.16 1.01 0.22 0.62 0.49 1.02 MET 42.88 0.82 4.21 441.49 43.94 1.27454.70 39.17 73.65 831.72 877.84 MHPG 0.46 0.51 0.55 0.42 0.44 0.48 0.610.95 0.66 0.40 MN 1.22 0.17 0.65 0.30 0.28 0.20 0.39 0.26 0.25 0.57 1.28NA5HT 0.14 0.17 0.11 0.14 0.21 0.17 0.17 0.18 0.12 NE 0.40 0.10 0.330.19 0.36 0.26 0.32 0.26 0.22 NMN 0.30 0.51 0.28 0.32 0.49 0.29 0.500.37 0.34 0.32 3.30 TPOL 8.59 6.11 7.77 9.11 11.33 13.89 16.47 36.251.19 1.41 28.63 TRP 186.72 27.57 7.44 240.92 47.34 5.07 53.65 55.253904.06 523.63 237.29 TRYPT 0.15 1.19 0.61 0.83 82.32 0.43 8.19 0.7413.85 0.50 277.80 TYR 266.09 551.51 251.17 717.54 98.95 140.30 214.95227.17 7411.08 1545.97 639.34 TYRA 579.81 10.61 0.19 0.90 525.82 2.03772.19 330.77 35.39 0.15 1088.90 URIC 695.51 703.83 3209.99 221.60 48.09580.64 229.01 405.36 314.90 1992.77 371.60 VMA 0.44 0.43 0.12 0.26 0.100.27 0.50 0.17 1.12 XAN 73.44 235.05 16.67 107.60 16.00 53.93 1458.7058.20 3669.12 343.39 45.54 XANTHOSINE 19.29 83.74 1.75 37.00 20.15 21.7823.70 16.64 1461.96 151.37 34.65 NORMALIZERS 8 OHdG pg/ml 11.2 3.33 2.228.47 10.8 4.9 17.39 2.42 635.48 0.32 1.61 80 h/lyrx 100 4.21 0.60 0.881.18 10.91 3.49 8.09 1.07 8.57 0.02 0.25 8 oh/cysx 100 15.93 4.59 4.0610.22 15.36 6.43 19.54 5.26 1273.76 1.48 3.32 8 oh/tyrax 1000 19.32313.84 11684.21 9411.11 20.54 2413.79 22.52 7.32 17956.49 2133.33 1.48

TABLE II CASE CODE 8 OH pg/gm CVL SAMPLES 8 OH*1000/tyr DIAG CODE cal byprotien 8 OH/O6 MG ESA006 17652.22 C 108934 0.12 ESA041 5198.99 C 87132ESA0447/21 3827.08 C 38897 ESA0447/16 3452.49 C 42132 0.24 ESA0512441.30 C3 44150 ESA037 1881.84 C3 31988 ESA011 1800.00 C NA ESA0481373.63 A, D, R 12012 ESA033R3 940.82 A, D, R 18821 1.33 ESA025CVL635.73 A, R 28812 ESA033R1 564.89 A, R 15168 ESA034 513.59 A, D, R 129970.92 ESA030 513.55 A, R 19281 ESA043 510.68 A, R 12113 0.77 ESA038502.68 A, R 23881 ESA040 215.30 A, R NA ESA050 197.87 A, R 10687 1.92esa032s 121.11 A, D, R 12188 esa029s 74.25 A, R 2188 ESA035s 38.50 A, D,R 1722 ESA018CVL 35.74 A, R 3221 ESA022CVL 27.73 A, R 3324 1.01 ESA00422.53 A, R NA ESA019CVL 22.05 A, D, R 1922 ESA003 20.53 AR 1334ESA023CVL 20.49 A, R 1987 ESA001 19.31 A, D, R 1332 ESA039 15.53 AR 1435ESA005 7.33 A 397 ESA036 3.79 A 433 esa027CVL 2.91 A 198 ESA047 1.56 N163 GT10 ESA012 1.48 N 289 ESA049 1.33 N 143 ESA020CVL 0.71 A 206esa031s 0.71 N 134 ESA048 0.64 N 289 ESA021CVL 0.81 A 189 7.44 ESA0420.57 N 215 ESA045 0.45 N 127 GT10 ESA026CVL 0.36 N 131 ESA024CVL 0.18 N156 CASE CODE PAP SMEAR SAMPLES 8 OH*1000/tyr DIAG CODE 8 OH pg/gm 8OH/O6 MG CON1R1 0.92 N 368 7.2 CON2 1.03 N 442 11.2 CON3 0.83 N 293 14.1CON4R1 0.72 N 528 NA CON1R5 endo brush 1.06 N 445 6.8 exo brush 0.94 N502 NA vaginal padl 0.12 N 28 NA CON5R1 2.44 N 308 11.1 CON4R3 0.98 N122 4.3 CON5R3 1.87 N 354 7.5 WOO1 2.19 A, R 125 1.25 W002 4.1 A, R 5070.73 W003 58.28 Hepatitis 3855 0.57 W004 8.23 A, R 200 0.58 W005 2.19 N75 1.58 W006 4.3 N, R 500 0.84 W007 2.1 N 90 0.71 W008 27.49 N, HPV 3610.55 W009 113.04 ?A, HPV 1300 0.45 W010 0.9 ?A 75 1.88 W011 1.6 N, R 751.2 W012 1.8 N 85 1.48 W013 1.1 N 75 1.2 W014 1.8 N, R 145 1.8 W015 1.9N 199 4.4 W016 2.8 N, R 301 4.3 W017 2.8 A, R 190 1.3 W018 3.12 A 4121.9 W019 1.4 N 138 7.3 W020 1.87 A 115 4.4 W021 4.12 N, R 543 1.7 W0221.7 A 214 GT10 W023 56.9 N, HPV 3224 0.41 W024 2.1 A, R 341 3 W025 0.81N 104 NA W026 2.3 N, R 122 5.7 W027 2.8 A, R 107 4.4 W028 1.6 N 115 8.1N—normal PAP, A—ASCUS, C—cancer, C3—CIN3, D—dysplasia, R—varios riskfactors, HPV—PCR+ viral te NA—not analysed, interference

TABLE III CVL SAMPLE DNA pm/um PLASMA URINE 8 OH*1000/ URINE ug/gm 8OH2′dG/ pg/ml urine code tyra 8 OH2′dG/creat 2dG 8 OH2′DG esa001u 3.15esa003u 4.97 esa004u 4.57 esa005u 17652.22 14.48 5.81 31.90 esa012u5198.99 3.30 16.30 esa006u 2441.30 5.73 esa018u 1881.84 2.38 6.13 22.10esa019u 1373.63 1.71 esa020u 940.82 4.11 835.73 2.86 4.98 14.60 esa022u564.89 3.94 5.87 esa023u 513.59 1.79 esa024u 513.55 4.83 esa025u 510.662.19 esa026u 502.68 2.22 esa027u 215.30 5.38 esa029u 197.87 4.14 esa030u121.11 2.16 esa031u 74.25 1.64 11.80 esa032u 38.50 3.25 esa033au 35.743.05 esa033bu 27.73 2.40 3.44 esa034u 22.53 2.15 3.38 15.30 esa035u22.05 3.04 esa036u 20.53 5.53 5.12 16.40 esa037 20.49 5.45 4.76 esa038u19.31 3.95 esa039u 15.53 3.55 esa040u 7.33 4.24 4.16 11.50 esa041u 3.792.13 4.28 esa042u 2.91 4.44 esa043u 1.56 3.64 esa0447/16u 1.48 3.33 3.36esa0447/21u 1.33 3.71 esa045u 0.71 3.24 12.30 esa046u 0.71 6.69 17.80esa047u 0.64 1.81 esa048u 0.61 2.58 esa049u 0.57 3.29 esa050u 0.45 2.50esa051u 0.36 4.11 esa011u 0.18 2.50

1. A method for determining the existence of infection in a body of anindividual living human subject comprising the steps of collectingsamples of cellular and extracellular material from at least oneselected site of the body of said individual subject, separating saidsamples into cellular and extracellular fractions, analyzing saidfractions for levels of 3-chlorotyrosine, and comparing the results ofsaid analyzing, wherein elevated levels of 3-chlorotyrosine in theextracellular fraction of the individual subject compared to thecellular fraction is indicative of infection.
 2. The method according toclaim 1, wherein the sampled material comprises stool samples.
 3. Themethod according to claim 1, wherein the sampled material comprisessaliva.
 4. The method according to claim 1, wherein the sampled materialcomprises urine.
 5. The method according to claim 1, wherein the sampledmaterial comprises phlegm.
 6. The method according to claim 1, whereinthe sampled material comprises cervico vaginal lavage or an endocervicalswab or brush sample.
 7. The method according to claim 1, wherein thesampled material comprises buccal or pharyngeal swab samples.
 8. Themethod according to claim 1, wherein the sampled material comprisesexfoliated urothelial cells.
 9. The method according to claim 1, whereinthe sampled material comprises alveolar or bronchial lavage.
 10. Themethod according to claim 1, wherein the sampled material comprises aPAP smear and endocervical swab or brush sample.
 11. The methodaccording to claim 1, wherein said levels of 3-chlorotyrosine aredetected electrochemically.